This project proposes to continue our studies on Carbohydrate Binding Protein 35, which has been renamed galectin-3 (Mr-30,000). The galectins comprise a family of galactose/lactose-specific saccharide-binding proteins that share characteristic amino acid sequences in the carbohydrate recognition domain of the polypeptides. Our interest in galectin-3 stemmed from the observation that the protein could be found in the nucleus, in the form of a ribonucleoprotein complex. Using nuclear extracts (NE) derived from HeLa cells, capable of carrying out splicing of pre-mRNA in a cell-free assay, depletion and reconstitution experiments demonstrated that galectin-3 was a splicing factor. In the course of these studies, we found another member of the galectin family, galectin-1 (Mr -14,000), was also detected in the nucleus and that it, too, served as a splicing factor. The activities of galectin-1 and galectin-3 in the splicing assay were redundant. A yeast two-hybrid screen has identified Gemin4 as an interacting ligand of galectin-1. Gemin4 is one component of a macromolecular complex, the SMN (Survival of Motor Neuron) complex, which has been visualized in subnuclear structures designated as gems. The function of the SMN complex is to supply snRNPs in the assembly of spliceosomes. Immunoprecipitation studies showed that the SMN complex contains both galectins-1 and -3. Thus, we now have in vivo evidence that galectins interact with a protein complex involved in splicing. On the basis of these and other observations, the specific objectives of the proposed research include: (1) to delineate and characterize the components and interactions of the SMN complex, on which Gemin4 and galectins-1 and -3 have been found; (2) to test for a role of galectins-1/-3 in the early steps of spliceosome assembly; (3) to test for an in vivo effect of galectin-3 and Gemin4 on pre-mRNA splicing; and (4) to continue our studies on the nucleo-cytoplasmic shuttling properties of galectin-3, in terms of import signals and in terms of a possible role in mRNA transport.